About 2% of all Americans are chronically infected with the hepatitis C virus (HCV), which eventually causes cirrhosis and liver cancer. HCV is the leading cause of liver failure in the USA and most liver transplant recipients are HCV positive. HCV infection is particularly common in HIV patients, where it can accelerate AIDS onset and lead to a myriad of complications for the immunocompromised patient. Current HCV therapies are costly and produce debilitating side effects. My laboratory studies the enzymes involved in the replication of the hepatitis C virus with a goal of finding new antiviral therapies. Recently we have developed a new high-throughput assay to screen for inhibitors of the viral NS3 helicase, which is needed for HCV RNA replication in human cells. Our goal is to find potential new drugs by identifying HCV helicase inhibitors with this new assay. The 50 5l assay has been optimized in 384-well microplates with a Z< factor of 0.7. In the reaction, a substrate consisting of a Cy5-labeled molecular beacon annealed to a longer DNA strand is pre-incubated with the HCV NS3 helicase and inhibitor. Upon initiation with 0.5 mM ATP, fluorescence is monitored for 30 minutes, after which time, control reactions are complete as measured by a complete loss of fluorescence. In reactions containing helicase inhibitors, fluorescence changes more slowly or not at all. Hits in this primary screen will be analyzed with DNA binding and RNA unwinding secondary screens to rule out compounds acting non-specifically. Specificity of NS3 helicase inhibitors will be further confirmed by analyzing compound effects on the NS3 protease and ATPase activities. Probes will then be used to explore the mechanism of the helicase reaction, to understand the role of the helicase in viral replication, and as leads for new antiviral drugs. PUBLIC HEALTH RELEVANCE: This project seeks to find new drugs to treat the virus that causes hepatitis C (HepC). Almost one in fifty Americans has been exposed to the HepC virus and over time it will cause progressive disease leading to liver failure in millions of Americans. The goal here is to find chemicals that prevent the virus from replicating in human cells. [unreadable] [unreadable] [unreadable]